Abstract

Background Defects in the posterior lamella of the eyelid are prevalent in the clinical settings, which comprises the tarsal plate and conjunctiva and has limited regenerative capacity. Currently, homogenous scaffolds are used for repair, but they fail to recapitulate the natural bilayer structure and reepithelialization. In this study, we designed a bilamellar scaffold (bGA-ADM) with a heterogeneous structure to repair such defects and promote the integrated regeneration of tarso-conjunctival composite tissue. Methods bGA-ADM was prepared by homogenization, freezing, freeze-drying, heat-vacuum crosslinking, and glutaraldehyde crosslinking, and its physiochemical properties were subsequently characterized. Fibroblasts, endothelial cells, and meibomian gland epithelial cells were seeded in the dermal layer, whereas mucosal epithelial cells were seeded onto the basement membrane layer to validate cytocompatibility and structural heterogeneity. Rabbit ear and eyelid implantation models were used to confirm in vivo biocompatibility and vascularization of bGA-ADM, and an eyelid posterior lamellar defect model was employed to evaluate its repairing efficacy. RNA sequencing was further performed to investigate the potential of bGA-ADM for meibomian gland construction. Results bGA-ADM exhibited biomimetic mechanical properties that provided adequate structural support, and allowed adaptive deformation. Consisting of a patterned basement membrane layer and a spongy dermal layer, it supported the stratified growth of fibroblasts, endothelial cells, meibomian gland epithelial cells, and mucosal epithelial cells in vitro. It also enhanced cellular infiltration and vascularization, thereby accelerating tissue regeneration in both ear and eyelid implantation models. When applied to repair the posterior lamellar defects in situ, bGA-ADM adaptively regenerated tissue closely resembling the native tissues, characterized by matrix deposition and conjunctival resurfacing. Furthermore